1. Field of Invention
This invention relates to a retrovirus inhibitor and more specifically to an HIV inhibitor.
2. Description of Related Art
There were an estimated 40.3 million people infected with HIV in 2005, with close to 5 million people becoming newly infected. AIDS is affecting an increasing number of women worldwide particularly in developing countries where transmission occurs primarily through heterosexual intercourse. A discrete, female-controlled method of preventing HIV infection is of paramount importance in the battle against AIDS. Since an effective vaccine is still years from development, topical microbicides have gained increased attention. Topical microbicides are vaginally or rectally applied compounds designed to inactivate HIV and prevent infection that should ideally be effective, safe, inexpensive and easy to administer. Microbicides, compounds that could be used in vaginal and rectal formulations, are increasingly seen as an urgent goal to stop transmission.
Advances in our understanding of the mechanism of HIV entry and infection have led to the development of microbicides that can target HIV without harming the body's natural defense system (ref 1-4). The initial, critical step of HIV infection is its entry through the fusion of the viral membrane with the membrane of either a T-cell or macrophage. The fusion process is mediated by the viral envelope glycoprotein, gp120, and can be triggered by interaction of gp120 with the T-cell antigen receptor CD4 glycoprotein (ref 5-7). CD4 induces conformational changes in gp120 that are postulated to promote subsequent steps in the fusion process, such as co-receptor binding and dissociation of gp120 from p41 (ref. 8-9). Several seven-transmembrane chemokine receptors, mainly CCR5 and CXR4, have been identified as obligate co-receptors for viral entry into the host cell (ref 9-13). Blocking the binding of CD4 with gp120 or preventing the CD4-induced conformational isomerization that promotes co-receptor binding and viral cell fusion could have potential value for the prevention and treatment of HIV infection and AIDS.
One candidate for a topical microbicide targeting gp120 is cyanovirin-N(CVN), an 11 kD protein originally isolated from the cyanobacteria Nostoc ellipsosporum (ref 14). It inactivates a broad range of clades of HIV-1, SIV, and FIV, and prevents cell to cell transmission of infection. Recent investigations using both in vitro and in vivo assays yield support for the efficacy of CV-N as a microbicidal candidate. Recombinant CVN blocked HIV-1 BaL infection of human ectocervical explants with no cytotoxic effects (ref 15). Gel formulations of CVN applied rectally to male macaques protected against challenge by the SIV/HIV-1 virus SHIV89.6P (ref 16). Further demonstration of in vivo efficacy was shown in a vaginal challenge model with female macaques. The macaques were treated with a vaginal gel containing CVN and challenged with SHIV89.6P. Under the challenge conditions of this assay, all placebo-treated and untreated controls (8 of 8) became infected, while 15 of 18 CVN treated macaques were not infected. CVN showed no clinically adverse effects in these in vivo assays.
CVN binds specifically to the highly glycosylated viral envelope protein gp120 and to the functionally analogous SIV proteins sgp130 and sgp140. In contrast, CVN does not bind appreciably to the soluble form of the cellular receptor CD4 (sCD4) or to a battery of other reference proteins. Investigations of CVN interactions by solution biophysical methods with both HIV-1 JRFL and HIV-1 89.6 envelope proteins gp120 and gp41 showed that the interaction of CVN with gp120 is of preferentially higher affinity, in the nM KD range, with a greater than 1:1 stoichiometry (ref 17). The epitopes on gp120 responsible for CVN binding appear to be predominantly high-mannose glycosylation sites of the Env, specifically terminal Man-α(1-2)manα-moieties on Man-8 and Man-9 glycans, and these appear key to the antagonist properties of the CVN molecule (ref 18-25). The high resolution structure of CVN has been solved by both X-ray crystallography and nuclear magnetic resonance spectroscopy (ref 26-30). These studies have identified carbohydrate binding sites on the CVN molecule. One of these appears to be higher affinity. Mutagenic analysis has shown that the high affinity site by itself is responsible for inhibition of HIV-1 fusion activity by CVN. Nonetheless, there are potential limitations, including amount of CVN production required based on measured in vivo efficacy, reliance on a single site of action and CV-N resistant strains of virus.
Another potential microbicide is the linear peptide 12p1 which was initially isolated from a phage display library and found to inhibit interaction of HIV-1 gp120 with both CD4 and a CCR5 surrogate, mAb 17b (ref 31). There is a direct interaction of 12p1 with gp120, which occurs with a binding stoichiometry of 1:1 (ref 32). The peptide was shown to inhibit the binding of monomeric YU2 gp120 to both sCD4 and 17b at IC50 values of 1.1 and 1.6 μM, respectively as determined by SPR analysis. This dual inhibition is a key feature of the action of 12p1. Peptide 12p1 also inhibited binding of these ligands to trimeric envelope glycoproteins, blocked the binding of gp120 to the native co-receptor CCR5, and specifically inhibited HIV-1 infection of target cells in vitro. Analyses of sCD4 saturation of monomeric gp120 in the presence or absence of a fixed concentration of peptide suggest that 12p1 suppression of CD4 binding to gp120 is due to allosteric inhibitory effects rather than competitive inhibition of CD4 binding. Using a panel of gp120 mutants that exhibit weakened inhibition by 12p1, the putative binding site of the peptide was mapped to a region immediately adjacent to, but distinguishable from, the CD4 binding footprint. 12p1 was unable to inhibit binding of sCD4 to a gp120 mutant, S375W, which is believed to resemble the CD4-induced conformation of gp120. The results obtained to date strongly suggest that 12p1 preferentially binds gp120 prior to engagement of CD4, and alters the conformational state of gp120 to a form that has suppressed interactions with receptor ligands (CD4 and CCR5/CXCR4) that are generally believed crucial for viral entry.
Thus, despite the foregoing developments, there is still a need in the art for a retrovirus inhibitor and more specifically for an HIV inhibitor.
All references cited herein are incorporated herein by reference in their entireties.